The following description provides a summary of information relevant to present disclosure and is not a concession that any of the information provided or publications referenced herein is prior art to the presently claimed invention.
Angiogenesis is the growth of new blood vessels from existing ones, and it is an important biological process for tissue development, growth, and repair. Angiogenesis is also an integral component of many physiological and pathological conditions such as wound healing, inflammation, and tumor growth (Folkman, J. and Klagsbrun, M. 1987. Science, 235: 442-447). Under abnormal conditions, angiogenesis can either directly or indirectly cause a particular disease that may include cancer, solid tumors, metastasis, diabetes, inflammation, cardiovascular disease, rheumatoid arthritis, psoriasis, inflammatory diseases, and Alzheimer's and Parkinson's diseases, and related neurological disease conditions, brain disorders, neurodegenerative disorders, neuropsychiatric illnesses, bipolar disorder, and diseases caused by aging. Angiogenesis may also exacerbate an existing pathological condition leading to other diseases, including eye retinopathies such as wet age-related macular degeneration, choroidal neovascularization, diabetic retinopathy, diabetic macular edema, retinal vein occlusion, and retinal angiomatus. These angiogenesis-dependent diseases are the result of new blood vessels growing excessively. In these conditions, new blood vessels feed diseased tissues and destroy normal tissues, and in the case of cancer, the new vessels allow tumor cells to grow and establish solid tumors or to escape into the circulation and lodge in other organs leading to tumor metastases.
There is considerable evidence showing that angiogenesis and chronic inflammation are closely related; the nature of this link involves both a considerable increase of cellular infiltration and proliferation, and the intervention of many growth factors and cytokines with overlapping activities (Jackson, J R et al. 1997, FASEB J, 11:457-465). Thus, targeting abnormal angiogenesis is important for the treatment of inflammation which is at the root of all chronic illnesses including but not limited to cancer, eye pathologies, diabetes, obesity, arthrosclerosis, reumathoid arthritis, heart, metabolic, skin, and brain diseases disorders, Alzheimer's, Parkinson's, Crohn's, pulmonary and bowel disease, dementia, depression, bipolar disorders, autism, to name a few. Inflammation, therefore, is a complex biological response of the vascular tissues (angiogenesis) to harmful stimuli such as cell damage, infections by pathogens, physical injuries, toxicants, irritants, foreign debris, burns, stress, and trauma.
Inflammation is a process by which the body's white blood cells and chemicals protect the body from infection and foreign substances such as bacteria and viruses. Inflammation can be acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and involves the vascular system, the immune system, the movement of blood cells and local cells into the injured tissues along with a cascade of biological events. When inflammation persists becomes chronic. There is stimulation of pro-inflammatory immune cells when they are not needed causing progressive damage to the cells and tissues (e.g., pancreatic tissues, gut mucosa, blood vessel lining, and joint tissue, to name a few) at the site of inflammation leading to a variety of diseases.
Many pro-angiogenic factors are mediators of inflammation (Campa et al. 2010, ID 546826, 1-14), and in some diseases, the body's immune system inappropriately triggers an inflammatory response when there are no foreign substances to fight off; in these autoimmune diseases, the body's normally protective immune system causes damage to its own tissues. Multiple sclerosis, type 1 diabetes mellitus, thyroiditis, rheumatoid arthritis, and lupus are autoimmune diseases. Thus, it is reasonable to deduce that most human diseases are inflammatory, and that this is mainly due to abnormal angiogenesis, defined as the uncontrolled growth of new blood vessels induced by the abnormal balance of many proteins involved in different cellular signaling pathways and biochemical functions in the body.
Numerous studies have demonstrated the direct association of abnormal angiogenesis, chronic inflammation and many human diseases. For example, inflammation triggered by microbes is a protective response against pathogens; however, it causes secondary damage to host tissues, i.e., DNA damage in various cell types resulting in carcinogenesis. Such inflammatory response induced by chronic infections with pathogens trigger liver, colorectal, and cervical cancers, and lymphoma (Kipanyula, M J. et al. 2012. Cell Signal, October 30. doi:pii: 50898-6568(12)00294-X. 10.1016/j.cellsig.2012.10.014). Therefore, chronic inflammation is a high risk for many cancers, including pancreatic cancer. For example, proteins such as nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are over-expressed in pancreatic cancer tissues; hyperlipidemia, obesity, and type II diabetes are also associated with chronic inflammation in the pancreas and involved in pancreatic cancer development (Takahashi M, et al. 2012. Semin Immunopathol. September 7 [Epub ahead of print]). It is clear from the examples that abnormal angiogenesis and inflammation play important roles in the pathogenesis of diseases.
Diseases of the eye are also closely related to angiogenesis and inflammation. Although there is not known lymphatic system in the eye, evidence from research studies have shown that the eye and their different surrounding tissues also have several lymphatic channels. Thus, both lymphangiogenesis and inflammation play important roles in pathological conditions in the eye including corneal transplant rejection, ocular tumor progression, macular edema, macular degeneration, choroidal neovascularization, among other abnormal conditions (Nakao S. et al. 2012, J. Ophthalmology. Article ID 783163, 11 pages doi: 10.1155/2012/783163).
The central nervous system (CNS) tissues, including the brain, the eye, and the spinal cord are protected from the circulation by a complex of biological barriers, and covered with a myeloid cell population known as microglia. When the CNS is damaged by acute insults, neurodegenerative conditions, and psychiatric disorders, there is an impairment of mechanisms such as neurogenesis and angiogenesis. This vascular dysfunction leads to cerebrovascular disorders, which cause neuropathological changes in the brain leading for example to dementia (e.g., Alzheimer's disease). Thus, cerebrovascular disease and microvascular alterations seem to interact with the underlying brain pathology, affecting the progression of cognitive deficits and encompassing changes in virtually all cell types of the neurovascular unit, including endothelial cells, vascular smooth muscle cells, pericytes, and astrocytes (Pimentel-Coelho P M and Rivest S. 2012. Eur J. Neurosci., 35(12):1917-37; Grammas P. et al, 2011. Int J Clin Exp Pathol. 15; 4(6):616-27).
Growth factors are capable of stimulating cellular growth, proliferation, and cellular differentiation and are involved in most cancers. They are important for regulating a variety of cellular processes and act as signaling molecules between cells (Welsh et al. Amer. J. Surg. 194, 2007, S76-S83). Excessive angiogenesis occurs when diseased cells produce abnormal amounts of growth factors or pro-angiogenic factors, overwhelming the effects of natural angiogenesis inhibitors. Pro-angiogenic growth factors include vascular endothelial growth factor (VEGF-A, B and C), fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF-a/b), epidermal growth factor (EGF), proepithelin (PEPI) or PC cell-derived growth factor (PCDGF) among many others (Marjon P L et al. Molecular Cancer 2004, 3:1-12; Kwabi-Addo B et al. Endocr Relat Cancer. 2004 11(4):709-24).
Cancer is caused by over-expression, and up-regulation of proteins (i.e., growth factors) implicated in many physiological pathways and endocrine functions, as well as the dysfunction of proteins that suppress cancer (i.e., p53) by interacting with other proteins (i.e., MDM2). When these cellular proteins become disfunctional, abnormal cells divide without control, and migrate and spread to any tissue through the blood and lymph systems (Hanahan D, Weinberg R A. 2000, Cell, 100(1):57-70) causing cancer. The most common cancers include breast, colon, pancreas, prostate, blood, bladder, brain, blood, bone, kidney, lung, liver, skin, ovarian, thyroid, gastrointestinal, head and neck, and neural, among others (Jemal et al. CA Cancer J. Clin. 2008, 58(2):71-96). Progress in cancer research has been slow since the drugs are mainly palliative and do not cure cancer; hence, there is a persisting need to develop effective therapeutic compounds that are more stable, more potent, with minimum or no toxicity, and that prolong the life of patients while providing significant improvement in their quality of life (QOL).
Pathological conditions of the eye include age-related macular degeneration, choroidal neovascularization, (AMD), proliferative diabetic retinopathy (PDR), diabetic macular edema (DME), among others. These diseases are the result of aberrant proliferation of new blood microvessels or neoangiogenesis (Hubschman et al. Clinical Ophthalmology 2009, 3 167-174). VEGF is a major factor in neovascular eye diseases and is the target of several anti-VEGF therapies based on monoclonal antibodies. Unfortunately, such therapies induce considerably side effects; thus, effective therapies are an unmet medical need.
Receptors, found in the extra cellular matrix, are transmembrane proteins that bind ligands. Integrins are receptors for a variety of extra cellular matrix proteins mediating migration of endothelial cells, and regulating their growth, survival, and differentiation, but there are also present on tumor cells of various origins (Cox et al, Nat Rev Drug Discov. 2010, 9(10):804-20). Receptors involved in human diseases include but are not limited to VEGF receptors, G protein receptors, ERBB receptors, platelet derived growth factor receptor (PDGFR), CXR1, CXR2, CCR3, CCR5 receptors, and NOGO receptors. Neurodegenerative diseases and mood disorders are example of diseases caused by the unbalanced neurotransmission of receptors and structural impairment of neuroplasticity. Chronic stress causes decrease of neurotrophin levels inducing depression. Antidepressants like lithium help increase expression of neurotrophins like BDNF and VEGF, thereby blocking, or reversing structural and functional pathologies via neurogenesis. Lithium also induces mood stabilization and neurogenesis due to the inhibition of glycogen synthase kinase-3beta (GSK-3beta), which allows the accumulation of beta-catenin. Increased levels of GSK-3beta and beta-catenin are associated with various neuropsychiatric and neurodegenerative diseases (Wada A. J Pharmacol Sci 2009, 110, 14-28). Thus, inhibition of GSK-3 beta expression seems therefore beneficial to ameliorate and/or stabilize mood disorders and induce neurogenesis.
The unbalanced presence of receptors also causes neurodegeneration. The Nogo receptor binds to the myelin-associated proteins Nogo-A, MAG, and OMgp, causing neurodegeneration. It can inhibit differentiation, migration, and neurite outgrowth of neurons, causing poor recovery of the adult central nervous system (CNS) from damage. Brain-derived neurotrophic factor stimulates the phosphorylation, suppressing Nogo-dependent inhibition of neurite outgrowth from neuroblastoma-derived neural cells; thus, it is important to control Nogo signaling to prevent neuronal damage.
Some proteins in the human body when suppressed exert a positive or beneficial effect. The target of rapamycin, mTOR, when inhibited suppresses the overexpression of HER2 oncoprotein, which is involved in cancer, or inhibits the process of aging by extending the lifespan of organisms (e.g., worms, fruit fly, yeast, and mice); mTOR, is therefore a suitable target to create potential anti-cancer and anti-aging compounds (Liu et al. Nature Reviews Drug Discovery 2009, 8:627-644). Other negative regulators of angiogenesis include thrombospondin-1, brain derived antiangiogenesis inhibitor, tumnstatin, angiostatin, somatostatin, tropomyosin, and endostatin among others. These proteins inhibit endothelial cell proliferation and tumor angiogenesis in vivo.
Diseases caused by pathogen agents include those acquired by blood borne pathogens like viruses (e.g., HIV, HCV, HBV, HSV, HTLV among others) through blood via infected people or animals, blood transfusions, or sexual contact. HIV/AIDS is a worldwide disease of large proportions (Richman, et al. Science 2009, 323, 1304-1307); yet there is no cure in spite of nearly four decades of basic and vaccine research.
Diseases caused by infectious agents include those caused by prions, which induce their own replication and derive from self; those caused by parasites (e.g., malaria, TB) acquired through bites by host organisms (e.g., insects, rodents), and those caused by pathogens acquired by contaminated food or water, or open wounds (e.g., bacteria, fungi, yeast).
Prions contain a protein (PrP) 27-30, which aggregates forming amyloid plaques that accumulate selectively in the central nervous system cells causing neurodegenerative diseases such as Creuzfeldt-Jakob and Alzheimer's diseases, Down's syndrome, fatal familial insomnia, and Parkinson's Disease. Prions are transmitted through contaminated plasma products, meat, and feeds or by person to person (Gu et al. JBC 2002, 277(3):2275-228). There are no drugs to treat prion infection, hence the need to develop novel drugs for this disease.
Bacterial and parasitic infections are a worldwide health problem. Staphylococcus aureus (MRSA) is a highly infectious bacteria and the cause of worldwide nosocomial infections. (Kaufmann et al., Exper. Opin. Biol. Ther. 2008, 8(6):719-724). Tuberculosis, caused by the pathogenic bacteria Mycobacterium tuberculosis (Mtb), is presently the leading cause of death from infectious disease, infecting more than a third of the world's population (Ciulli et al. Chem Bio Chem 2008, 9, 2606-2611). It is acquired from small-infected mammals or by person to person. Salmonella typhimurium, other highly infectious and deadly bacteria, spreads by drinking contaminated water (Townes et al. Biochemical and Biophysical Research Communications 2009, 387: 500-503). Malaria, caused by the protozoan Plasmodium falciparum, is spread by mosquito bites infecting the red blood cells (VanBuskirk et al. PNAS, 2009, 106(31):13004-13009).
The diseases described above are the result of the abnormal balance of many proteins involved in different functions and physiological pathways in the body. Thus, it is clear that many different signaling proteins and biochemical pathways are involved in abnormal angiogenesis and chronic inflammation and many proteins that are abnormally over-expressed or down-regulated trigger such abnormal angiogenesis and inflammation. Drugs approved to treat many of these diseases are single target drugs that provide a modest and transient clinical effect, but do not cure the aimed disease, and most are non-specific. Furthermore, clinical trials of drugs targeting many of these diseases have shown numerous times that targeting a single protein or an angiogenesis pathway or a single mechanism, or a single disease condition, is unlikely to result in the best possible benefit for the patient; clinical trials with combination therapies, for cancer, (i.e., chemo, radiation, and antibodies), or HIV (HAART), to name a few, have proven unsuccessful since none of these approaches cure either cancer or HIV infection. Therefore, there is an urgent and persisting need to develop novel and unique multi-targeted therapies.
It would be therefore advantageous to create therapeutic compounds carrying a plurality of different synthetic stereoisomer peptides in their retro-inverso or inverso and linear and cyclic configuration for the purpose of simultaneously and independently targeting different pathologic proteins involved in a disease. This approach may allow simultaneous interference at different levels in the biochemical cascade, or interference of different cellular pathways that lead to disease when proteins are abnormally over-expressed or down regulated. For example, targeting simultaneously several proteins involved in abnormal angiogenesis and inflammation would enable therapeutic applications for cancer, eye pathologies, brain diseases, neurological diseases, diabetes, cardiovascular diseases, arthritis, infectious diseases, psoriasis, Alzheimer's and Parkinson's diseases, diabetes, bipolar disorders, among many others.
Accordingly, there is need to create novel and unique compounds by searching, finding, integrating, converging, modifying, and applying existing knowledge and technologies. This invention precisely follows such approach to create for the first time novel and unique ligand-targeted multi-stereoisomer peptide-polymer conjugate compounds that can be used as therapeutics for the treatment of a variety of human diseases. The particular medical application of a therapeutic compound created in this invention, will depend on the plurality of specific and unique stereoisomer peptides comprised in the polymer conjugate.
A variety of methods described in the literature to synthesize peptides, are aimed at improving, modifying or providing alternative approaches for their synthesis, and for the terminal groups protection, and coupling that can be applied depending on the structure of the peptide to be synthesized, and the conformation. Such peptide synthesis methods are well known to those of skill in the art (see Stewart J M and Young J D, 1984, Solid phase peptide synthesis (2nd ed.). Rockford, Pierce Chemical Company; Atherton E and Sheppard R C, 1989, Solid Phase peptide synthesis: a practical approach. Oxford, England: IRL Press; and Henklein et al, 2008, J. Peptide Science 14 (8): P10401-104; Greene's Protective Groups in Organic Synthesis, 4th ed., John Wiley & Sons, Inc., 2007). Methods for synthesizing retroinverso peptides, which are similar to their L-counterparts, may also vary depending on the sequence of the stereoisomer peptide, their configuration and structure (see Briand et al. 1997, PNAS 94:12545-50, and Venkataramanarao et al. 2006, Tetrahedron Letters 47: 9139-9141). However, none of these methods provide any detail as to how synthesize, and chemically modify the stereoisomer peptides of this invention.
Cyclization of stereoisomer peptides to create cyclo peptides is an important feature of this invention. Peptides containing several Cys residues in the core of the peptide or at the ends of each side of a linear peptide form disulfide bonds which are achieved by using a variety of oxidation reactions. Similarly, methods of peptide cyclization that do not form disulfide bonds but rather create other type of bonds through linking of the terminal residues of the peptide, or the side chains of residues in the peptide, are also available, and are known to those of skill in the art (see Methods of cyclization are described by Bulaj G and Olivera B M, 2008, Antioxid Redox Signal, 10(1):141-55, and Amit M et al, 2009. Biochemistry, 48 (15):3288-3303). However, none of these methods describe the actual cyclization of stereoisomer peptides, with D-amino acids, and in the retroinverso or inverso configuration. Hence, there is the need to make modifications to create ands describe procedures that include the stereoisomer peptides of this invention.
To be able to effectively deliver drugs inside tissues or cells, a variety of polymers such as PLGA, PCL, HPMA, PEG, have been used because they produce tailored surface properties with specific physical, chemical, and biological properties that are suitable for medical applications. However, the selective delivery of therapeutic agents by polymers to disease tissue or cells in vivo remains a major challenge since it depends on the particular physicochemical properties of the polymer and the drug (see Zhang, Y and Chu C C. 2002, J. Biomater. Appl. 16: 305-325, and Liu J et al., 2004, J. Pharm. Sci. 93: 132-143) and the biological pathway for delivery (see Qaddoumi M G et al. 2003. Mol. Vis., 9: 559-568). In spite of this complexity, polymers have been used in a variety of medical and biotechnological applications for controlled delivery of small molecules (mainly cytotoxic) and large biomolecules (proteins and antibodies) inside tissues or cells (see Jeong B et al. 1997, Nature 388: 860-862; Bae Y H et al. 1997. Ann. N.Y. Acad. Sci. 831: 47-56, and Zhao et al. 2003, Adv. Drug Deliv. Rev., 55:483-499). These methods, however, have never been used to carry a plurality of different stereoisomer peptides, and none of them have described the conjugation or encapsulation of a plurality of stereoisomer peptides in their retroinverso and cyclic configuration. In this invention, for the first time such techniques with modifications are applied to create the novel therapeutic compounds of this invention.
The synthesis of low and high molecular weight oligomeric forms of polymers such as lactide and glycolide and their applications as carriers for drug delivery was carried out several decades ago (see Lewis D H. 1990. Controlled release of bioactive agents from lactide-glycolide polymers. In: Chasin M, Langer R, editors. Biodegradable polymers as drug delivery systems. New York: Marcel Dekker, p: 1-41, and Wu X S. 1995. Synthesis and properties of biodegradable lactic/glycolic acid polymers. In: Wise et al. Eds. Encyclopedic Handbook of Biomaterials and Bioengineering. New York: Marcel Dekker, p:1015-10541). These polymers are FDA approved and have wide acceptance in surgical procedures due to their biocompatibility and biodegradation through cleavage of its backbone ester linkages (see Tice T R and Cowsar D R. 1984. Pharm Technol, 11:26-35). However, none of these methods have described the conjugation or encapsulation of a plurality of stereoisomer peptides in their retroinverso and cyclic configuration with polymer PLGA. This invention presents the creation of such novel PLGA based therapeutic compounds.
Methods for encapsulation of drugs using a variety of size particles or carriers have also been described. The encapsulation of drugs entails the formation of polymer particles of a variety of sizes including nanoparticles, microparticles, miliparticles, nanocapsules, microcapsules, milicapsules, nanoemulsions, microemulsions, nanospheres, microspheres, and those made of a variety of substances to obtain liposomes, oleosomes, vesicles, micelles, surfactants, phospholipids, sponges, and those made with cyclodextrines. Thus, particulated polymers such as microspheres, microcapsules, and nanoparticles, are very useful because they can be administered by different routes in vivo (see Jain R A, 2002, Biomaterials, 21: 2475-2490; and Berkland C et al., 2002, J. Control Release, 82: 137-147). Polymer nanoparticles are used to encapsulate the novel polymer conjugates created in this invention.
Drugs of any size, regardless of molecular weight and solubility, can be loaded in the biodegradable microparticles using different manufacturing techniques. They include emulsion polymerization, interfacial polymerization, solvent evaporation, salting out, coacervation, combination of sonication and layer by layer technology, and solvent displacement/solvent diffusion mong others. Each method of drug encapsulation requires its own specific condition for stability, solubilization, and control releases immune-elimination (see Rajiv A J. 2000, Biomaterials, 21: 2475-490, and Sinha V R and Trehan A. 2003. J. Control. Release, 90:261-280). The method of encapsulation, therefore, is entirely based on the physicochemical activity of the type of drug and its intended application. Here specific modification and combination of methods are used to create the nanoparticles loaded with the composition of matter of this invention.
Another polymer amply used in biomedical applications is HPMA due to its biocompatibility and high solubility in water. HPMA has been conjugated mainly to low molecular weight drugs to increase their therapeutic effect and reduce their toxicity (e.g., toxic cancer drugs); these conjugates have also been labeled with fluorescent or radiolabeled tags to analyze the biodistribution of the drug-HPMA conjugate in tissues and cells. The selection of HPMA for biomedical applications relies on its extensive research, well-known chemical and structural properties, and their suitability as carriers for drug delivery, especially of toxic anti-cancer molecules, in many clinical applications (see U.S. Pat. No. 5,037,883; Kopecek, et al, Eur. J. Pharm. Biopharm., 2000, 50: 61-81; Vicent M J et al. 2008. Expert Opin Drug Deliv. 5(5):593-614; Greco F and Vicent M J. 2008. Front Biosci. 2008 13:2744-56). Methods to synthesize HPMA to produce HPMA copolymers, the characterization of their properties, and the preparation of conjugates are standard and well established in the art (see Europ. Polym. J. 9, 7, 1973; Europ. Polym. J. 10 405, 1974), but none of these methods have ever been used to create the novel compounds of this invention.
In sum, the methods described above for peptide synthesis, their modification, and their conjugation to polymers or encapsulation have never been used to create the novel ligand-targeted multi-stereoisomer peptide-polymer conjugate compounds of this invention. Therefore, this invention for the first time uses such approaches with variations to create the novel therapeutic compounds described in this specification together with the examples, their representation in the figures and the claims describing the particular characteristics of the novel compounds. These compounds also provide targeted specificity to treat a particular disease and ideal biopharmaceutical properties that make them highly stable and suitable for any route of administration.
In view of the forgoing, it is appreciated that these novel and unique ligand-targeted multi-stereoisomer peptide-polymer conjugate compounds for a variety of therapeutic interventions, constitute a significant advancement in the art, and a new approach to treat human diseases.